The applicant cloned a homeobox gene called Six3 which, when overexpressed in fish embryos, causes ectopic lens formation. This has obviously inspired the applicant to pursue this gene further as one which is very important in embryonic vertebrate eye development. The goal of the proposal is to understand the role of Six3 during vertebrate eye development and to elucidate the regulatory pathways involved in the process. The applicant has already made mice which have the Six3 gene inactivated via embryonic stem cell methods. The proposal is to study these mice further. The applicant has already identified at least some of the regulatory elements of Six3 and proposes to study the evolutionary conservation of these elements between vertebrates and flies. The molecules that control Six3 expression in the anterior neural plate in optic vesicles will also be identified using the characterized regulatory elements as probes to screen a mouse E8.0-8.5 anterior neural plate expression library. Targets regulated by Six3 will be revealed by subtraction of cDNAs prepared from wild type in Six3-/anterior neural plate/eye field embryonic regions. Novel regulatory genes that participate in the process of eye development will be identified by the construction of a cDNA library specifically derived from the Six3 expressing cells in the anterior neural plate and eye field of E8.0-8.5 mouse embryos. These experiments will further define the mechanisms and pathways involved in vertebrate eye development and should identify novel genes controlling early steps of this process. Thus, they will enhance our understanding of eye-related birth defects.